Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.     

Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at −196 °C in liquid nitrogen with 10% Me₂SO as cryoprotectant for 24 h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10 mM sodium β-glycerophosphate, 50 μM l-ascorbic acid, and 10 nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.     

Hypoxia enhances the viability,growth and chondrogenic potential of cryopreserved human adipose-derived stem cells

Cryopreservation is the only existing method of storage of human adipose-derived stem cells (ASCs) for clinical use. However, cryopreservation has been shown to be detrimental to ASCs, particularly in term of cell viability. To restore the viability of cryopreserved ASCs, it is proposed to culture the cells in a hypoxic condition. To this end, we aim to investigate the effect of hypoxia on the cryopreserved human ASCs in terms of not only cell viability, but also their growth and stemness properties, which have not been explored yet. In this study, human ASCs were cultured under four different conditions: fresh (non-cryopreserved) cells cultured in 1) normoxia (21% O₂) and 2) hypoxia (2% O₂) and cryopreserved cells cultured in 3) normoxia and 4) hypoxia. ASCs at passage 3 were subjected to assessment of viability, proliferation, differentiation, and expression of stemness markers and hypoxia-inducible factor-1 alpha (HIF-1α). We found that hypoxia enhances the viability and the proliferation rate of cryopreserved ASCs. Further, hypoxia upregulates HIF-1α in cryopreserved ASCs, which in turn activates chondrogenic genes to promote chondrogenic differentiation. In conclusion, hypoxic-preconditioned cryopreserved ASCs could be an ideal cell source for cartilage repair and regeneration.     

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of −150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.     

Cryopreservation of adipose-derived stromal/stem cells using 1–2% Me2SO (DMSO) in combination with pentaisomaltose: An effective and less toxic alternative to comparable freezing media

Mesenchymal stromal/stem cells (MSCs) derived from bone marrow, umbilical cord and especially adipose tissue are increasingly being explored for their therapeutic potential to treat a wide variety of diseases. A prerequisite for most allogeneic off-the-shelf and some autologous MSC therapies is the ability to safely and efficiently cryopreserve cells during production or for storage prior to treatment. Dimethyl sulfoxide (Me₂SO) is still the commonly used gold standard cryoprotectant (CPA). However, undesirable cellular impacts and side effects of Me₂SO have led to an increasing demand for the development of safe and effective alternatives.This study investigated the effect of pentaisomaltose as a CPA for cryopreservation of adipose-derived stromal/stem cells (ASCs). We compared pentaisomaltose-based freezing media containing 1% Me₂SO (PIM1) or 2% Me₂SO (PIM2) to our in-house freezing media formulation containing 10% Me₂SO (STD10) and to CryoStor freezing media containing 2% or 10% Me₂SO (CS2 and CS10). We assessed the recovery of viable ASCs, their phenotype, differentiation potential, proliferation potential, and migratory potential. Further, their immunomodulatory potential was assessed by measuring their ability to suppress T cell proliferation and express immunomodulatory markers.The results showed that the post-thaw viability of ASCs cryopreserved with STD10, CS10 and PIM2 was improved compared to that of CS2. The recovery of ASCs with PIM1 and PIM2 was also improved compared to that of CS2. Proliferation and migration were comparable among the tested freezing media. The results showed no difference in the induction of PDL1, PDL2 or IDO1 expression. Nevertheless, the potential of cryopreserved ASCs to suppress T cell proliferation was reduced when the Me₂SO concentration was reduced (CS10>STD10>CS2 and PIM2>PIM1).Altogether, the migratory and immunomodulatory potential combined with improved recovery indicate that the addition of pentaisomaltose in the freezing media may allow for the reduction of the Me₂SO concentration to 2% while retaining a more potent cell product that what is recovered using comparable freezing media. With the desire to reduce the amount of Me₂SO, these results suggest that 2% and potentially even 1% Me₂SO in combination with 10% pentaisomaltose could be an effective and less toxic alternative to comparable freezing media.     

Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix

Cryopreservation of tissue engineered products by maintaining their structure and function is a prerequisite for large-scale clinical applications. In this study, we examined the feasibility of cryopreservation of tissue engineered bone (TEB) composed of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs) and partially demineralized bone matrix (pDBM) scaffold by vitrification. A novel vitreous solution named as VS442 containing 40% dimethyl-sulfoxide (DMSO), 40% EuroCollins (EC) solution and 20% basic culture medium (BCM) was developed. After being cultured in vitro for 8 days, cell/scaffold complex in VS442 was subjected to vitreous preservation for 7 days and 3 months, respectively. Cell viability, proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons being made with those cryopreserved in VS55 vitreous solution. Compared with that cryopreserved in VS55, cell viability and subsequent proliferative ability of TEB in VS442 after being rewarmed were significantly higher as detected by live/dead staining and DNA assay. The level of alkaline phosphatase (ALP) expression and osteocalcin (OCN) deposition in VS442 preserved TEB was also higher than those in the VS55 group since 3 days post-rewarm. Both cell viability and osteogenic capability of the VS55 group were found to be declined to a negligible level within 15 days post-rewarm. Furthermore, it was observed that extending the preservation of TEB in VS442 to 3 months did not render any significant effect on its survival and osteogenic potential. Thus, the newly developed VS442 vitreous solution was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB over VS55.     

Stromal stem cells from adipose tissue and bone marrow of age‐matched female donors display distinct immunophenotypic profiles

Adipose tissue is composed of lipid‐filled mature adipocytes and a heterogeneous stromal vascular fraction (SVF) population of cells. Similarly, the bone marrow (BM) is composed of multiple cell types including adipocytes, hematopoietic, osteoprogenitor, and stromal cells necessary to support hematopoiesis. Both adipose and BM contain a population of mesenchymal stromal/stem cells with the potential to differentiate into multiple lineages, including adipogenic, chondrogenic, and osteogenic cells, depending on the culture conditions. In this study we have shown that human adipose‐derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) populations display a common expression profile for many surface antigens, including CD29, CD49c, CD147, CD166, and HLA‐abc. Nevertheless, significant differences were noted in the expression of CD34 and its related protein, PODXL, CD36, CD 49f, CD106, and CD146. Furthermore, ASCs displayed more pronounced adipogenic differentiation capability relative to BMSC based on Oil Red staining (7‐fold vs. 2.85‐fold induction). In contrast, no difference between the stem cell types was detected for osteogenic differentiation based on Alizarin Red staining. Analysis by RT‐PCR demonstrated that both the ASC and BMSC differentiated adipocytes and osteoblast displayed a significant upregulation of lineage‐specific mRNAs relative to the undifferentiated cell populations; no significant differences in fold mRNA induction was noted between ASCs and BMSCs. In conclusion, these results demonstrate human ASCs and BMSCs display distinct immunophenotypes based on surface positivity and expression intensity as well as differences in adipogenic differentiation. The findings support the use of both human ASCs and BMSCs for clinical regenerative medicine. J. Cell. Physiol. 226: 843–851, 2011. © 2010 Wiley‐Liss, Inc.     

Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells

Bone tissue engineering(BTE) is now a promising re-search issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self re-newal and multi-lineage differentiation. Unlike embry-onic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells(BMSCs) are the ear-liest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its' clinical ap-plication. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stemcells(ASCs), is found to be more suitable in clinical ap-plication because of high stem cells yield from lipoaspi-rates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated be-cause most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation poten-tial. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in-vivo research reviews revealed more controversies in this issue. We expect the new researchers can have a quick understanding of the progress in this filed and design a more comprehensive research based on this review.     

Long bone mesenchymal stem cells (Lb-MSCs): clinically reliable cells for osteo-diseases

Mesenchymal stem cells (MSCs) have been designated as the most reliable cells in clinics to treat osteo-diseases because of their versatile nature. MSCs, isolated from long bone (Lb-MSCs) are rarely reported and named as RIA-MSCs because of the reamer–irrigator–aspirator (RIA) device. The potential of these cells in the treatment of non-union bone fractures made them the ideal candidates to be studied for clinical practices. In this work, effect of cryopreservation on the proliferation and differentiation capabilities of long bone MSCs (Lb-MSCs) has been studied. For this purpose, Lb-MSCs were isolated via RIA device and characterized using flow cytometry and differentiation assays. Cells were cryopreserved for 3, 6 and 12 months and thereafter were characterized using differentiation assays and genetic markers specific for osteogenic, chondrogenic, and adipogenic potential quantitatively by qRT-PCR. Lb-MSCs were found expressing MSC characteristic markers defining their identity. The population doubling time (PDT) was about 2.5 ± 0.5 days and colonies appeared after 7–10 days. Differentiation potential and gene expression of 3, 6 and 12 months cryopreserved Lb-MSCs were unaltered. The results show that cryopreservation did not have an effect on the differentiation potential of human Lb-MSCs. Therefore, our work offers Lb-MSCs as clinically cells for treating osteo-diseases.     

Isolation,characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models

One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adipose-derived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous pre-clinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.     

Adipose-derived stem cells: An appropriate selection for osteogenic differentiation

Mesenchymal stem cells (MSCs) are a major component of various forms of tissue engineering. MSCs have self-renewal and multidifferential potential. Osteogenic differentiation of MSCs is an area of attention in bone regeneration. One form of MSCs are adipose-derived stem cells (ASCs), which can be simply harvested and differentiated into several cell lineages, such as chondrocytes, adipocytes, or osteoblasts. Due to special properties, ASCs are frequently used in vitro and in vivo bone regeneration. Identifying factors involved in osteogenic differentiation of ASCs is important for better understanding the mechanism of osteogenic differentiation. Different methods are used to stimulate osteogenesis of ASCs in literature, including common osteogenic media, growth factors, hormones, hypoxia, mechanical and chemical stimuli, genetic modification, and nanotechnology. This review article provides an overview describing the isolation procedure, characterization, properties, current methods for osteogenic differentiation of ASCs, and their basic biological mechanism.     

Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold

Recent studies have shown that liposuction aspirates from rat, rabbit, mouse, and human sources contain pluripotent adipose tissue-derived stromal cells (ASCs) that can differentiate into various mesodermal cell types, including osteoblasts, myoblasts, chondroblasts, and preadipocytes. To develop a research model for autologous bone tissue engineering, we isolated ASCs from human liposuction aspirates (hASCs) and induced their osteogenic differentiation in three-dimensional poly(dl-lactic-co-glycolic acid) (PLGA) scaffolds. Human liposuction aspirates were proteolytically digested and centrifuged to obtain hASCs. After primary culture in control media and expansion to three passages, the cells were seeded in two-dimensional plates or three-dimensional PLGA scaffolds and cultured in osteogenic media for 4 weeks. In two-dimensional culture, osteogenesis was assessed by RT-PCR analysis of the osteogenic-specific bone sialoprotein mRNA, by alkaline phosphatase staining, and by von Kossa staining. In three-dimensional culture, osteogenesis was assessed by von Kossa and alizarine red S staining at 1, 2, and 4 weeks following osteogenic induction. hASCs incubated in two-dimensional osteogenic media stained positively for alkaline phosphatase and with von Kossa stain after 2 weeks of differentiation. Expression of the osteogenesis-specific bone sialoprotein gene was detected by RT-PCR after 2 weeks of differentiation. PLGA scaffolds seeded with hASCs showed multiple calcified extracellular matrix nodules by von Kossa and alizarine red S staining after 2 weeks of differentiation. In conclusion, the authors identified an osteogenic potential of hASCs and demonstrated osteogenic differentiation of hASCs into an osteogenic lineage in three-dimensional PLGA scaffolds.     

Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies

The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 ± 1.1%, mean ± SEM) and hip/thigh region (1.2 ± 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.     

The effects of β-tricalcium phosphate 3D scaffold in-situ cryopreservation on the migration rate and osteogenic ability of mesenchymal stem cells

To readily supply seed cells for tissue engineering and ensure their constant availability for experiments, it is imperative to establish an in-situ cryopreservation method for cell storage. We investigated the effects of a β-tricalcium phosphate (β-TCP) 3D scaffold in-situ cryopreservation method on the migration rate and osteogenic ability of mesenchymal stem cells (MSCs). Compared to using a 2D plate culture and trypsinized cryopreservation, MSCs on β-TCP 3D scaffolds demonstrated a higher amplification rate, and the harvest and survival rates (HSR) increased from 55.9 to 81.3% when the 3D in-situ cryopreservation method was applied. The cell migration rate and alkaline phosphatase (ALP) activity were unaffected after in-situ cryopreservation, and unexpectedly, the Specific ALP activity of migrating cells was higher than that of non-cryopreserved cells, suggesting that the cell-scaffold combination could be cryopreserved using the present protocol without loss of proliferative or osteogenic potential. These findings highlight a methodology for 3D scaffold in-situ cryopreservation and passage for MSC production in bone tissue engineering, and present the possibility of designing a perfusion cells/scaffold factory for scale-up production.     

Activation and promotion of adipose stem cells by tumour necrosis factor‐alpha preconditioning for bone regeneration

There is a major medical need for developing novel and effective approaches for repairing non‐union and critical‐sized bone defects. Although the mechanisms remain to be determined, it is known that inflammation plays a crucial role in initiating bone repair and regeneration. This study investigated the effect of short‐term (3 days) preconditioning with tumor necrosis factor‐alpha (TNF‐α) on proliferation, mobilization, and differentiation of adipose tissue‐derived mesenchymal stem cells (ASCs). We demonstrated that TNF‐α pre‐conditioning increased proliferation, mobilization, and osteogenic differentiation of ASCs and up‐regulated bone morphogenetic protein‐2 (BMP‐2) protein level. BMP‐2 silencing by siRNA partially inhibited osteogenic differentiation of ASCs induced by TNF‐α; BMP‐2 pre‐conditioning also significantly increased osteogenic differentiation of ASCs but the effects were significantly smaller than those observed for TNF‐α preconditioning. Furthermore, TNF‐α treatment promoted extracellular‐signal‐regulated kinases(Erk)1/2 and p38 mitogen‐activated protein kinase (MAPK) signaling pathways, but only Erk1/2 inhibition reduced the BMP‐2 levels and osteogenic differentiation induced by TNF‐α preconditioning. Together, these results support the hypothesis that inflammation contributes to bone regeneration by promoting proliferation, mobilization, and osteogenic differentiation of ASCs; 3 days of TNF‐α preconditioning, mimicking the short boost of inflammation normally occurring after bone injury, might serve as a feasible approach for directing stem cells into osteogenic differentiation. J. Cell. Physiol. 9999: XX–XX, 2013. © 2013 Wiley Periodicals, Inc. J. Cell. Physiol. 228: 1737–1744, 2013. © 2013 Wiley Periodicals, Inc.     

Bone regeneration in a rabbit ulna defect model: use of allogeneic adipose-derivedstem cells with low immunogenicity

Tissue engineering provides new potential treatments for the repair of bone defects. Bone-marrow-derived mesenchymal stem cells (BMSCs) represent an attractive cell source for therapeutic applications involving tissue engineering, although disadvantages, such as pain of harvest and low proliferation efficiency, are major limitations to the application of BMSCs in the clinic. Adipose-derived stem cells (ASCs) with their multilineage potential and satisfactory proliferation potential can be induced into the osteogenic lineage in vitro and can be anchored onto suitable scaffolds as seed cells to repair bone defects successfully in an autologous setting. Previous studies have indicated that both undifferentiated BMSCs and ASCs exhibit immunosuppression and immunoprivilege properties. We compare the immuno-function of undifferentiated and osteo-differentiated ASCs in vitro and explore the feasibility of applying allogeneic ASCs to the repair of ulnar bone defects in the rabbit model. Our study demonstrates that allogeneic osteogenic differentiated ASCs maintain low immunogenicity and negative immunomodulation. The allogeneic osteogenic differentiated ASCs combined with demineralized bone matrix successfully regenerate ulnar bone defects in rabbits without immunosuppressive therapies.     

Cells isolated from cryopreserved dental follicle display similar characteristics to cryopreserved dental follicle cells

Dental follicle tissue is a promising resource of mesenchymal stem cells for cytotherapeutic approaches and tissue engineering applications. There are two procedures for banking of human dental follicle stem cells have been reported. Conventional method requires cell isolation, expansion and immediate cryopreservation. Whereas dental follicle stem cells can be isolated from cryopreserved dental follicle fragments. The aim of this study was to compare the characteristics of dental follicle cells isolated from cryopreserved fragments (DFCs-CF) with dental follicle cells recovered from cryopreserved cells (DFCs-CC). Dental follicle fragments obtained after mechanical disaggregation were divided into two parts, with one part maintained in culture, while another part underwent cryopreservation. Dental follicle fragments and dental follicle cells from fresh tissue were stored in liquid nitrogen for 3 months. After thawing, the isolation, morphology, proliferation, cell cycle, colony-forming-unit ability, stemness-related marker expression, apoptosis, and multi-lineage differentiation potential of DFCs-CF were tested compared with DFCs-CC. DFCs-CF expressed mesenchymal stem cells marker, proliferated well, showed similar levels of mRNA for stemness- and apoptosis-related genes and exhibited the capacity of multi-lineage differentiation similar to those of DFCs-CC. These results imply that cryopreservation of dental follicle fragments is an effective banking method for isolation of dental follicle cells.     

Adipose-derived stem cells retain their regenerative potential after methotrexate treatment

In musculoskeletal tissues like bone, chemotherapy can impair progenitor cell differentiation and proliferation, resulting in decreased bone growth and mineralization throughout a patient?s lifetime. In the current study, we investigated the effects of chemotherapeutics on adipose-derived stem cell (ASC) function to determine whether this cell source could be a candidate for repairing, or even preventing, chemotherapy-induced tissue damage. Dose-dependent proliferation rates of ASCs and normal human fibroblasts (NHFs) were quantified after treatment with cytarabine (CY), etoposide (ETO), methotrexate (MTX), and vincristine (VIN) using a fluorescence-based assay. The influence of MTX on the multipotency of ASCs and freshly isolated stromal vascular fraction (SVF) cells was also evaluated using lineage-specific stains and spectrophotometry. ASC and NHF proliferation were equally inhibited by exposure to CY and ETO; however, when treated with MTX and VIN, ASCs exhibited greater resistance. This was especially apparent for MTX-treated samples, with ASC proliferation showing no inhibition for clinically relevant MTX doses ranging from 0.1 to 50 μM. Additional experiments revealed that the differentiation potential of ASCs was not affected by MTX treatment and that upregulation of dihydrofolate reductase possibly contributed to this response. Moreover, SVF cells, which include ASCs, exhibited similar resistance to MTX impairment, with respect to cellular proliferation, clonogenicity, and differentiation capability. Therefore, we have shown that the regenerative properties of ASCs resist the cytotoxicity of MTX, identifying these cells as a potential key for repairing musculoskeletal damage in patients undergoing chemotherapy.     

Boron increases the cell viability of mesenchymal stem cells after long-term cryopreservation

The field of stem-cell biology has emerged as a key technology for the treatment of various disorders and tissue regeneration applications. However, a major problem remains in clinical practice, which is the question of whether stem cells preserve their self-renewal and differentiation potential in the culture conditions or not. In the current study, effects of boron on the cryopreservation of human tooth germ stem cells (hTGSCs) were evaluated for the first time. The impacts of various boron concentrations (sodium pentaborate pentahydrate (NaB)) were tested on characterized hTGSCs viability for different time intervals (24, 48, and 72 h). 20 μg/ml NaB with lower Me₂SO concentration was found to display positive effects on hTGSCs during repeated freezing and defrosting cycles, and long-term cryopreservation. After thawing, cells were analyzed for their surface antigens and differentiation capacity. hTGSCs were successfully cryopreserved without any change in their mesenchymal stem cell characteristics as they were treated with boron containing freezing medium. In addition, fatty acid composition was examined to demonstrate membrane fatty acid profiles after freeze-thawing. Besides, NaB treatment extended osteogenic and chondrogenic differentiation of hTGSCs remarkably after long-term cryopreservation with respect to control groups. The study clearly suggests that NaB has a protective role on the survival of hTGSCs in short- and long-term cryopreservation. Due to the possible storage of hTGSCs at early ages, development of a functional and reliable cryopreservation media can be designed as a future solution to the dental stem cell banking.     

Cryopreservation of hMSCs seeded silk nanofibers based tissue engineered constructs

Long term cryopreservation of tissue engineering constructs is of paramount importance to meet off-the shelf requirements for medical applications. In the present study, the effect of cryopreservation using natural osmolytes such as trehalose and ectoin with and without conventional Me₂SO on the cryopreservation of tissue engineered constructs (TECs) was evaluated. MSCs derived from umbilical cord were seeded on electrospun nanofibrous silk fibroin scaffolds and cultured to develop TECs. TECs were subjected to controlled rate freezing using nine different freezing solutions. Among these, freezing medium consisting of natural osmolytes like trehalose (40 mM), ectoin (40 mM), catalase (100 μg) as antioxidant and Me₂SO (2.5%) was found to be the most effective. Optimality of the chosen cryoprotectants was confirmed by cell viability (PI live/dead staining), cell proliferation (MTT assay), microstructure analysis (SEM), membrane integrity (confocal microscopy) and in vitro osteogenic differentiation (ALP assay, RT-PCR and histology) study carried out with post-thaw cryopreserved TECs. The mechanical integrity of the cryopreserved scaffold was found to be unaltered.